N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the  Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors that includes EGF, transforming growth factor (TGF)-α, amphiregulin, betacelulin, and neuregulin Abnormal development of the brain is implicated in the etiology and/or pathology of various psychiatric diseases, including schizophrenia At the present time, little is known about the relationship between HB-EGF and psychiatric disorders. In the current study, therefore, we have generated HB-EGF conditional knockout (KO) mice that exhibit disruption of HB-EGF activity specifically in the ventral forebrain. We have compared the neurobehavioral and molecular biological characteristics of these mice with those of control mice and have demonstrated that the HB-EGF KO mice exhibit behavioral and biological abnormalities similar to those described in various psychiatric disorders. These results strongly suggest that alterations in HB-EGF signaling contribute to the pathogenesis of mental illness.Hb-egf gene causes lethality Hb-egf gene by lacZ staining. LacZ positive cells were founded in the prefrontal cortex and hippocampus in KO mice  . HoweverTo investigate the possible role of HB-EGF in sensorimotor gating, prepulse inhibition (PPI) was measured in HB-EGF KO and control mice. Sensorimotor gating is the neural process in which allow attention to be focused on one stimulus. Sensorimotor gating can be assessed as PPI of startle, which is the modulation of the startle response, following a weak prepulse. In the pulse-only trials, startle amplitude did not differ significantly between HB-EGF KO and control mice, . HB-EGF KO mice showed diminished PPI at prepulse intensities of both 73 and 76 dB (P<0.05 vs. control mice) . MoreoveTo assess the social affiliative behavior of HB-EGF KO mice, we employed a social interaction paradigm. When introduced to another mouse, a normal mouse will typically investigate the new mouse by walking over and sniffing it. But animal models of psychiatric disorders tend to show decreases in such social-interaction behavior. During a 10 min social interaction test in a novel environment, the mean duration per contact was significantly less in HB-EGF KO mice than in control mice, indicating that HB-EGF KO mice spent significantly less time engaging in social contact (P<0.05 vs. control mice) . We coulHb-egf gene on learning and memory performance. The exploratory behavior of the HB-EGF KO mice and control mice did not differ during the training session (P>0.05 vs. control mice)  . Howeverol mice) . In contol mice) .Next, to further assess the specificity of changes in working memory related to the loss of HB-EGF protein, we elected to test spontaneous alternation in the Y-maze, which is devoid of any procedural bias because it is based on the natural tendency of mice to explore a novel environment. There was no difference in the number of arm choices or in preference for a particular arm between HB-EGF KO and control mice . HoweverHb-egf gene affected the function of monoaminergic neuronal systems, tissue levels of monoamines and their metabolites were assessed in various regions of the brain in HB-EGF KO and control mice. In the prefrontal cortex, DA, 5-HT, and its metabolite 5-hydroxyindole acetic acid (5-HIAA) levels were statistically lower in HB-EGF KO mice than in control mice (P<0.05 vs. control mice)  . The 5-Hol mice) . In contol mice) . On the ol mice) .To try to identify possible morphological correlates of the observed abnormalities of behavior and cognition, we analyzed the morphology of pyramidal neurons in cortical layer III in the prefrontal cortex. Next, we assessed by western blotting the protein levels of NR1 and PSD-95 as the obligatory subunits of the NMDA receptor and its interacting molecule, which are thought to be involved in the pathophysiology of schizophrenia. By using western blotting, we next evaluated the protein levels of total CaMKII α and β, phosphorylated CaMKII (p-CaMKII) α and β, p21-activated kinase (p-PAK)1/3, and p-PAK2, which are serine-threonine kinases that regulate the synaptic architecture in neurons. To investigate the mechanisms that might lead to the observed behavioral abnormalities and altered monoamine levels, we compared the expression of the EGF receptor and its signaling in HB-EGF KO and control mice. No significant differences in the expression of total EGF receptor or ErbB4 proteins were observed between controls and HB-EGF KO mice (P>0.05 vs. control mice) . DespiteIn this study, we generated conditional HB-EGF KO mice using a Cre/loxP system and were able to confirm the disruption of HB-EGF in the prefrontal cortex and hippocampus of KO mice. These results corresponded with a previous report which detected the Six3 expression in the ventral forebrain and basal ganglion To investigate the roles of HB-EGF in psychomotor behavior, we performed a comprehensive behavioral analysis of ventral forebrain-specific HB-EGF KO mice. Deficiencies in PPI and memory tasks are considered to be signs of cognitive dysfunction HB-EGF KO mice also displayed deficit of memory related behavior, as observed by the Y-maze test performance and novel-object recognition test. These deficits are considered to be signs of cognitive and working memory dysfunction, respectively Hb-egf gene might be expected to result in hypoplasia and hypofunction of dopaminergenic neurons. On the other hand, the upregulation of NE, 5-HT, and its metabolites observed in the striatum of the KO mice in the present study may compensate for the downregulation of the prefrontal monoamine levels.In general, abnormal behaviors are accompanied by altered monoamine levels in animal models of psychiatric disorders The presence of NMDA receptor hypofunction in psychiatric disorder has been inferred recently from a large number of clinical and preclinical observations The most significant finding made in the present study was that the HB-EGF KO mice had a significantly decreased spine density on the primary apical dendrites of the layer III pyramidal neurons of the prefrontal cortex (vs. control mice) of approximately 40%. Since the dendritic spine is the major site of excitatory synaptic transmission in the central nervous system, the size and density of these spines will clearly influence the operation of functional circuits Hb-egf cDNA) demonstrated that a mutation of the Hb-egf gene was associated with decreases in the activation of EGF receptor and ERK Additionally, current evidence indicates that cytokines and growth factors can strongly influence neural phenotypic differentiation and subsequent neural function. Some studies have indicated a potential link between impaired EGF signaling and the pathogenesis of schizophrenia Hb-egf gene, could affect synaptic plasticity by regulating NDMA relating proteins, and this would lead to the abnormal behaviors observed in HB-EGF KO mice.MAPKs, also known as ERKs, operate in a signaling cascade that regulates various cellular processes . ERKs are recognized as being important signaling components in learning and memory and in regulating dendritic spine density In conclusion, the current study demonstrates that HB-EGF KO mice exhibited the behavioral abnormalities reflected in a comprehensive spectrum of psychomotor and cognitive dysfunctions, similar to many psychiatric disorders. Based on the observations from the current study, we proposed a role for HB-EGF in determining monoaminergic neural system and synaptic plasticity. Many psychiatric disorders are multifactorial, reflecting longitudinal and complex interactions of causative agents, including genetic and environmental factors. These findings indicate that the HB-EGF signaling may play a pivotal role in the pathogenesis of a number of psychiatric disorders, including schizophrenia.The targeting strategy has been described previously Mice  were anesthetized with sodium pentobarbital , and the brains were perfusion-fixed with 4% paraformaldehyde  in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). The brains were removed from the mice after 20 min of perfusion fixation at 4°C and then immersed in the same fixative solution. Brain sections were equilibrated in 25% sucrose solution and quickly frozen in Tissue-Tek O.C.T. .Hb-egf (NM_010415) nucleotide positions 546 to 665 were synthesized, in situ hybridization analysis was performed, as previously reported For lacZ staining, after fixation with 0.2% glutaraldehyde and 1% formalin, tissues were incubated with 5-bromo-4-chloro-3-indoly-β-galactoside   for 2 hr at 4°C. For immunohistochemical staining, frozen sections were washed for 5 min in 0.01 M PBS and then treated with 0.3% hydrogen peroxidase in 10% methanol. They were then washed three times in 0.01 M PBS, followed by a 30 min pre-incubation with 10% normal goat serum. They were then incubated with a rat anti-HB-EGF antibody (1∶200), including 0.3% triton X-100, overnight at 4°C. After a 15 min rinse in changes of 0.01 M PBS, the sections were incubated with a secondary antibody, Alexa 488-conjugated goat anti-rat IgG . In situ hybridization analysis was performed using an antisense DIG-labeled cRNA probe. DIG-labeled cRNA probes corresponding to All mice  were housed in a room with a 12-hr light/dark cycle (light on at 8:00 a.m.) and had ad libitum access to food and water. Behavioral tests were performed between 9:00 a.m. and 5:00 p.m., except for the 24-hr locomotor activity test. All procedures relating to animal care and treatment conformed to the Animal Care Guidelines of the Animal Experiment Committee of Gifu Pharmaceutical University.To measure the locomotor activity, a mouse was placed in the cage, . Locomotion was measured every 1 hr for 1 day using digital counter with infrared sensor . Animals were placed into the cages at 2:00 P.M. for a 24 period. The room light was on from 8:00 A.M. to 8:00 P.M. Vehicle (0.3% tartaric acid), clozapine , or haloperidol  was administrated once a day for 9 days, and one day after the final injection, locomotor activity test was started.Acoustic startle responses were measured in a startle chamber  using standard methods described previously The social interaction test in a novel environment was done in a manner similar to published method Mice were tested in a circular arena, 40 cm in diameter. All mice were habituated in the apparatus (without objects) for 40 min per day prior to the training session. At the end of each trial, the animals were removed from the arena and the arena was cleaned with 70% ethanol solution and dried with paper toweling. Object recognition was scored by the amount of time spent for each object, with the nose of the mouse directed to and located within 2 cm from the object or with the anterior limbs touching the object. In a first trial (T1: 5 min) two similar objects (left and right: circle) were placed in a symmetrical position 5 cm away from the wall. In a second trial (T2: 5 min), two plastic dissimilar objects [one with a circle (left) and a new one triangle (right)] were presented. The number of account exploring each object during T1 and T2 was recorded. All mice were tested 1 hr between T1 and T2.Recording the spontaneous alteration behavior in Y-maze was assessed as spatial reference memory task. The apparatus consisted of three identical arms (50 l×16 w×32 h cm) made by black Plexiglas. Each mouse was placed at the end of one fixed arm and allowed to move freely through the maze during an 8 min session. The sequence of arm entries was recorded manually. An alternation was defined as entries into all three arms on consecutive choices. The number of maximum alternations was then the total number of arms entered minus two, and the percentage of alternation was calculated as  ×100. In addition, the total number of arms entered during the session was also determined.Each mouse  was decapitated under deep anesthesia and its brain quickly removed from the skull, briefly washed in ice-cold saline, and laid on a cooled (4°C) metal plate, on which it was rapidly dissected to separate the prefrontal cortex, striatum, cerebellum, and thalamus. The dissected brain regions were weighed, frozen, and stored at −80°C until analysis. The regional levels of monoamines and their metabolites were determined using a high-performance liquid chromatography (HPLC) system equipped with an electrochemical detector . The HPLC system and conditions were as follows: Eicom EP-300/Eicom ECD-300 system; column, Eicompak SC-5ODS (3.0 mm i.d.×15 cm) with precolumn; mobile phase, 83% 0.1 M citric acid/0.1 M sodium acetate buffer, 17% methanol, 0.18% sodium 1-octanesulfonate, containing 5 mg/ml disodium-EDTA; flow rate, 0.5 ml/min; electrode, Eicom WE-3G graphite electrode; reference electrode, Eicom RE-100 Ag/Ag Cl; applied voltage, 750 mV versus Ag/Ag Cl. Column temperature was maintained at 25°C.Mice were anesthetized with pentobarbital and perfused via the ascending aorta with phosphate-buffered saline  until the outflow became clear, followed by 0.1 M phosphate buffer  containing 4% paraformaldehyde (PFA) for 15 min. Brains were removed and postfixed in the same fixative for 24 h at 4°C. The brain was sliced at 150 µm thickness using a vibratome . The slices were transferred into a Silgard-coated Petri dish filled with 4′, 6-diamidino-2-phenylindole (DAPI) solution  in PBS. Neurons in the infralimbic (IL) and prelimbic (PL) regions of the medial prefrontal cortex (mPFC) were penetrated with glass microelectrodes filled with 3% LY  in distilled water, and LY was injected by passing 2–20 nA current with 2.5 second pulses applied at 0.2 Hz for 1–2 minutes. After LY injections, sections were postfixed in the same fixative for 1–2 days, and then were incubated as follows: several washes in PBS, 1 h in 0.1 M PBS containing with 1% BSA, 0.3% TritonX-100 and 0.1% NaN3 (blocking solution); and with a rabbit anti-LY polyclonal antibody  in blocking solution at 4°C for 4–5 days. After washing, sections were incubated for 3 h with Alexa 488-labeled anti-rabbit IgG . After several washes in PBS, sections were mounted on slides with Vectashield (Vector Laboratory). Immunofluorescent images were analyzed using a confocal laser scanning microscope (Leica TCS). Pyramidal neurons within the infralimbic and prelimbic regions were defined by the presence of a single apical dendrite. For analyses of branching, basal dendrites were evaluated by counting the number of branches on each dendrite. Data were expressed as the mean number of branches per dendrite. The average spine density (number of spines per 10 µm of dendritic length) was estimated on the first segments of apical dendrites. For analyses of spine density and spine length, spines were identified on the basis of the morphological criteria for ‘mushroom’ and ‘thin’ spines described by Peters and Kaiserman-Abramof Immunoblotting analyses of the prefrontal cortex region were performed using male, 10-week-old animals (controls and KO mice). Extracts prepared from the medial prefrontal cortex region were treated with Laemmli's sample solution and boiled for 3 min. Samples containing equivalent amounts of protein were separated on 10% sodium dodecyl sulfatepolyacrylamide gels and transferred for 2 hr at 70 V to a polyvinylidene difluoride membrane using the method of Towbin et al Immunoblotting analyses of the autophosphorylation of the ErbB receptor were performed using the prefrontal cortex region. In this study, membrane protein was extracted using a Native Membrane Protein Extraction Kit  according to the manufacturer's instructions. Then, membrane protein was immunoblotted using an anti-phospho-EGF receptor antibody and an anti-phospho-ErbB4 antibody , as described above.P-value of <0.05 was considered to be statistically significant.All data were expressed as the mean±SEM. Statistical significant was evaluated either by either one-way or two-way ANOVA test followed by a post hoc Dunnett's test. A Figure S1Supporting figures(0.16 MB DOC)Click here for additional data file.Table S1Supporting Table(0.04 MB DOC)Click here for additional data file.Table S2Supporting Table(0.02 MB DOC)Click here for additional data file.


The aim of this study was to detect abnormally expressed microRNA (miRNA) in keloids and to study their functions. The differential expression of miRNAs in keloids and normal tissue was detected by gene microarray. MiRNA expression was verified by real-time PCR. A luciferase reporter gene assay, western blot, and real-time PCR were used to detect the effect of miR-194-3p on RUNX2. An MTT assay and a transwell assay were used to detect the effect of miR-194-3p in both primary cultured fibroblasts and HKF cells. Related proteins were analysed by western blot and real-time PCR. The expression of miR-194-3p was lower in keloids, and MiR-194-3p was shown to target RUNX2 directly. MiR-194-3p inhibited the proliferation and migration of fibroblasts through the inhibition of CDK4 and MMP2. MiR-194-3p and RUNX2 may become new targets for the prevention and treatment of keloids. The formation of a keloid seriously impacts the appearance of an individual; the symptoms vary to different degrees and can cause great harm to the physical and mental health of the patient . The preMiRNAs (MicroRNAs) are endogenous noncoding RNAs with lengths of approximately 18 to 22 nucleotides. Various physiological and pathological processes have been shown to be associated with the abnormal expression of miRNAs. Recent studies have demonstrated that many miRNAs may play pivotal roles in keloid scarring by regulating the proliferation, apoptosis, and metastasis of fibroblasts . StudiesWe aimed to evaluate the expression levels of microRNAs in keloid fibroblasts and explore their regulatory roles in keloids. We hope that our research will provide new possibilities for the prevention and treatment of keloids.A total of 28 cases of keloid and corresponding normal skin tissue were derived from the General Hospital of North Theater. PLA between January 2015 and February 2017. All keloids were confirmed by clinical or pathological examination, the course of the disease was longer than 1 year, and patients had not received treatment. The specimens were removed and stored in liquid nitrogen. The experimental study was approved by the ethics committee of the General Hospital of Shenyang military region, and all patients were informed and provided consent.HKF (human keloid fibroblast) cells obtained from the Shanghai cell bank of the Academy of Sciences were the primary culture of keloid fibroblasts. Cells were cultured in a 5% CO2 atmosphere at 37°C in DMEM containing 10% foetal bovine serum. The keloid tissue was minced and placed in pancreatic enzyme digestion at 37°C for 30 min. Then, DMEM containing 10% foetal bovine serum was added to the medium, terminating the reaction. Three to five generations of cells were used for these experiments.Total RNA was extracted from cells using the mirVana™ miRNA isolation kit , and RNA concentrations were measured using a NanoDrop® ND-1000 spectrophotometer . RNA with an A260/A280 ratio of nearly 2.0 was considered to be high quality RNA.A microarray analysis was performed on RNA samples (3 paired) under microgravity and under static conditions in replicates. Expression data for each sample were obtained on the Affymetrix GeneChip Human Primeview Array. Hybridization was carried out for a duration of 16 h at 60 rpm at 48°C, and slides were scanned on the GeneChip microarray Scanner 3000 7G. Raw data were extracted after the slides were scanned, and raw datasets were analysed using GeneSpring GX 12.6 software, followed by differential gene expression (DE), fold-change, and cluster analysis.http://www.mirdb.org/) and targetscan (http://www.targetscan.org). Each target gene and its corresponding regulatory loci were obtained using the input target miRNA.The miRDB database software was entered through a network search of miRDB , and the reaction ran for 40 cycles. The expression of miR-194-3p was detected by stem-loop primers. The results were analysed with ABI Prism 7500 SDS software. The primers used are shown in The primary cultures of keloid fibroblasts and HKF cells were divided into two groups. RUNX2 wild type/mutant type (wt/mut) and miR-194 mimic/control/antisense (AS) were transfected into cells. RUNX2 mut is a plasmid in which the binding site of RUNX2 to miR-194-3p is mutated. Twenty-four hours after the transfection, precooled lysate and the reaction solution were added. A luminescent meter was used to read the fluorescence value and analyse the data. The plasmids and RNA fragment we used were purchased from Shanghai Shenggong company .μM nitrocellulose membranes . Membranes were blocked with Tris-buffered saline plus 0.2% Tween 20 (TBS-T) containing 3% BSA , followed by overnight incubation with the primary antibody and washing with TBST buffer. Then, membranes were incubated with the secondary antibody , diluted in blocking buffer for 1 h at room temperature, and washed again with TBS-T. Antibody-reactive proteins were detected with enhanced chemiluminescence, Amersham ECL Plus western blotting detection reagents . RUNX2, CDK4, MMP2, and GAPDH antibodies were purchased from Santa Cruz Biotechnology (USA).Cells and tissues were lysed with radio immunoprecipitation assay (RIPA) buffer, mixed with Laemmli sample buffer (1×), and boiled. Proteins were subjected to 10% SDS-PAGE and electroblotted onto 0.22-4 cells/well. After incubation for 24 h, the miR-194 mimic/control or miR-194 inhibitor/control was transfected into fibroblasts. Following the transfection, 5 mg/mL MTT was added at 0, 12, 24, 36, and 48 h. After 4 h of culture, the OD value at 490 nm was measured.The effect of miR-194 on cell proliferation was detected by MTT assay. The cells were inoculated in 96-well culture plates at a density of 1 × 105/ cells were inoculated in the upper chamber with 200 μL of medium. A total of 600 μL of medium were added to the lower chamber of the 24 wells. Twenty-four hours after the transfection, 95% ethanol was used to immobilize cells, and trypan blue was added. The number of cells was calculated by photographing and counting the cells under a microscope.The effect of miR-194 on cell migration was detected by transwell assay. A total of 1 × 10We constructed miR-194-3p overexpressing stable cell lines by transfecting cells with the miR-194-3p mimic and then selected with puromycin (1.5mg/ml). The miRNA mimic negative control was transfected as a control.We constructed miR-194-3p low expression stable cell lines by transfecting cells with the miR-194-3p inhibitor and then selected with puromycin (1.5mg/ml). The miRNA inhibitor negative control was transfected as a control.To stably silence RUNX2, cells were transfected with the si-RUNX2 fragment (Shanghai GeneChem Company), and a random fragment was used as a control. After 3 weeks, stable cells were selected, cultured, and amplified.μl pipette tip across the cell monolayer. Cells were rinsed with PBS and cultured in the medium. Wound closure was detected at 0 and 24 h and imaging performed under a microscope.Wound scratch assays were performed to detect cell migration. HKF cells were seeded in 6-well plates and allowed to reach confluence. An artificial wound was made using a 200  P values less than 0.05 were considered statistically significant.All experiments were repeated three times. SPSS, version 19 software, was used for statistical analysis, with data presented as x±s (standard deviation). Statistical significance was determined with unpaired Student t tests, except as noted for analyses of microarray data, which were examined with Fisher exact tests.The results of the chip detection revealed that many miRNAs, including miR-194-3p, miR-451, and miR-144-5p, were differentially expressed in keloids compared with adjacent tissue . These rAll 28 cases of keloid tissue we collected were tested by real-time PCR. The results showed that miR-194-3p has lower expression in keloid tissue . Using mAfter transfecting the miR-194-3p mimic or inhibitor into cells, the MTT assay revealed that miR-194-3p significantly inhibited the proliferation of cells. When miR-194-3p was inhibited, the proliferation of cells increased significantly Figures .CDK4 is a cell cycle-related protein downstream of the RUNX2 pathway . ThroughThe results of the transwell assay showed that the overexpression of miR-194-3p significantly inhibited the migration of cells . When miP<0.05)  RNA significantly suppressed cell proliferation and cell migration ( Figures . The inh Figures . These rmiRNAs are a class of endogenous noncoding small RNA molecules that have regulatory and control functions on genes. Such noncoding small RNA molecules play an important role in processes including biological development, metabolism, differentiation, proliferation, and apoptosis. Previous studies have indicated that abnormal expression of miRNAs can cause disease and even cancer, as certain miRNAs function similarly to tumour suppressor genes , 13. KelIt was shown that miRNAs are involved in the regulation of fibroblast proliferation, differentiation, and extracellular matrix synthesis. In addition, a complex functional network is formed between miRNA and keloids , 15. It We found that there are many differentially expressed miRNAs in keloids compared with adjacent tissues. Among these differentially expressed miRNAs, we showed for the first time that the expression of miR-194-3p in keloids was significantly lower compared with normal tissue. Real-time PCR revealed that miR-194-3p was downregulated in keloid tissues. The lower miR-194-3p expression was significantly correlated with family genetic history. However, due to the difficulty of sample collection, the sample size of this study is relatively small, which is a limitation of our study. To study the effect of miR-194-3p on keloids, miRDB and targetscan software were used to predict the target genes of miR-194-3p. Further analysis revealed that the 3′ UTR region of miRNA-194-3p has a binding site leading to the direct regulation of the expression of RUNX2.RUNX2 is a major transcription factor regulating organ formation, and it affects the proliferation and migration of many solid tumours . RUNX2 wStudies showed that downregulation of RUNX2 expression significantly inhibited cell proliferation, migration, and invasion of cells . We founStudies have shown that RUNX2 can improve the migratory ability of various cells . The traIn conclusion, our study revealed that miR-194-3p significantly inhibits the proliferation and migration of fibroblasts. The inhibitory effect of miR-194-3p on fibroblasts may be achieved through the targeting effect of miR-194-3p on RUNX2. MiR-194-3p may become a potential target for the prevention and treatment of keloids.


Here, for the first time, we discuss the biomimetic inclusion of both these sequences within a self-assembled minimalistic peptide hydrogel. Here, based on our work with Fmoc-FRGDF , we show it is possible to present two epitopes simultaneously via the assembly of the epitopes by the coassembly of two SAPs, and compare this to the effectiveness of the signals in a single peptide; Fmoc-FRGDF: Fmoc-PHSRN (N-flourenylmethyloxycarbonyl-proline-histidine-serine-arginine-asparagine) and Fmoc-FRGDFPHSRN . We show both produced self-supporting hydrogel underpinned by entangled nanofibrils, however, the stiffness of coassembled hydrogel was over two orders of magnitude higher than either Fmoc-FRGDF or Fmoc-FRGDFPHSRN alone. In-vitro three-dimensional cell culture of human mammary fibroblasts on the hydrogel mixed peptide showed dramatically improved adhesion, spreading and proliferation over Fmoc-FRGDF. However, the long peptide did not provide effective cell attachment. The results demonstrated the selective synergy effect of PHSRN with RGD is an effective way to augment the robustness and functionality of self-assembled bioscaffolds.Self-assembling peptides (SAPs) are a relatively new class of low molecular weight gelators which immobilize their solvent through the spontaneous formation of (fibrillar) nanoarchitectures. As peptides are derived from proteins, these hydrogels are ideal for use as biocompatible scaffolds for regenerative medicine. Importantly, due to the propensity of peptide sequences to act as signals in nature, they are easily functionalized to be cell instructive via the inclusion of bioactive epitopes. In nature, the fibronectin peptide sequence, arginine-glycine-aspartic acid (RGD) synergistically promotes the integrin α The self-assembly of biomolecules have received significant attention as a facile route to fabricate bioinspired scaffolds with porous, three-dimensional 3D) architectures, that have increasingly well-understood mechanisms to control their formation ,2,3. NevD archite5β1 receptor [n); RGDG13PHSRN within a polyethylene glycol hydrogel [5RGDSP) in a self-assembling peptide-amphiphile (PA) [In nature, the scaffold responsible for this level of control over cell fate is the tissue-specific extracellular matrix (ECM); a multifunctional apparatus that provides structural support and dynamic cellular signaling to control a range of cellular functions, such as cell adhesion, differentiation, migration and proliferation . Generalreceptor ,18. Recereceptor ,20, as wreceptor ,21,22 anreceptor . These shydrogel  or a serile (PA) . These mile (PA) . Hydrogeile (PA) . The mecile (PA) . Systemsile (PA) , and witile (PA) . We repoIn this work, we designed three peptide sequences containing one or both of the synergistic bioactive epitpopes: Fmoc-FRGDF, Fmoc-PHSRN and Fmoc-FRGDFPHSRN. The self-assembly mechanism, mechanical properties, micro- and nano-structures of the peptide scaffolds were evaluated and compared, and the synergistic effect of peptides within the scaffolds was assessed by monitoring the adhesion, spreading and proliferation of human mammary fibroblast cells (HMFCs).The synthesis of all Fmoc-peptides was performed as previously reported . Purity The required amount of Fmoc-peptides was suspended into 400 µL Mili-Q water . 0.5 M NaOH  was added dropwise to the aqueous suspension of the peptides until fully dissolved. During this time, the peptide solutions were placed in an ultrasonicator  and vortexed interchangeably. A required volume of 0.1 M HCL was then added drop wise until physiological pH (7.4) was reached with vortexing. Finally, 0.1 M phosphate buffer saline (PBS) (pH 7.4) was added to the peptide solution to bring total volume up to 1 mL and maintain pH, then kept at room temperature for gelation .Information on secondary structure of the Fmoc-peptides was obtained by CD spectra using a Jasco J-815 spectropolarimeter using a 1-mm path length quartz cell . Measurements were carried out in continuous scanning mode, at a scan rate of 50 nm/min with a bandwidth of 1 nm and 2 s integration time. Reported spectra were averages of 10 scans with data-smoothing, collected over a range of wavelengths from 190 to 300 nm. Sample was diluted 20 times using Mili-Q water to give a peptide concentration of 0.05 mg/mL. Mili-Q water was used as a background and subtracted from all spectra.FT-IR spectra were collected on a Nicolet 6700 FTIR spectrophotometer  in attenuated total reflection (ATR) mode. 0.1 M PBS was used as a background before samples measurement. 12 µL of peptide hydrogel was spread directly on the ATR crystal and allowed to dry for 10 min evaporating the solvent which enables minimal contribution form the solvent to the spectra. The samples were scanned between the wavenumbers of 4000 and 400 cm −1 over 64 scans. Data analysis was carried out using OPUS software .Fluorescence emission spectra were analysed on a Cary Eclipse fluorescence spectrophotometer . Samples were diluted using Mili-Q water to give a final peptide concentration of 0.5 mg/mL. A quartz cuvette of 1 mm path length  was used. Excitation wavelength were at 250 nm and emission data were collected between 300 and 600 nm with a data pitch of 1.0 nm and a scanning speed of 600 nm/min.AFM images were obtained using a Multimode 8 AFM  in peak force QNM  mode. Samples were diluted 20 times using Mili-Q water to make a final concentration of 0.05 mg/mL. A 15 µL aliquot of the diluted solution was deposited onto a highly ordered pyrolytic graphite (HOPG) substrate (SPI), the excess sample solution was absorbed by pipettes and left to dry overnight. Scanasyst-air silicon-nitride tip (Bruker) with spring constant of 0.4 N/m was used. AFM scans were taken at 512 × 512 pixels resolution and the topographic images of the samples were captured at a scan rate of 0.93 Hz with scan size of 10 µm.TEM visualization was operated on a JEOL-2100 LaB6 transmission electron microscopy  connected to a Gatan Orius CCD camera  at an operation voltage of 100 kV. For effective penetration of electron beam, the hydrogel sample was diluted 5 times (peptide concentration 0.2 mg/mL) and 12 µL droplet of diluted solution was placed on an agar lacey carbon coated film on 300 mesh copper grids  and allowed to adsorb for 30 s, the excess fluid was blotted down using Whatman filter paper (No. 1). In negative staining, the carbon side of the grid was stained with one drop of NanoVan . After 5 min, the excess fluid was removed from the grid and dried in air for 2 min with the carbon side up; lastly the grid was put into grid box to leave it dry overnight.q) to be measured across the range of 0.01 to 0.6 Å−1. The diffraction pattern was recorded on a Pilatus 1M detector , and processed using the Australian Synchotron ScatterBrain Software . Hydrogels were prepared as detailed above two days prior and loaded into 1.5 mm sealed glass capillaries. PBS backgrounds were collected before samples were loaded. Each triplicate sample (and background) was subjected to three 5-s exposure times at multiple positions along the capillary. Repeat measurements were summed using Scatterbrain and q calibrated using an AgBeh sample. The intensity was normalized and set on an absolute scale using water and air shots. Due to poor scattering, backgrounds were scaled by 0.9 prior to subtraction from the sample scattering data. The data was then subjected to indirect Fourier transform (IFT) analysis and P(r) inversion using SASView  to calculate the average diameter of the fibrils in the sample.SAXS was performed using the SAXS/WAXS beamline at the Australian Synchrotron . Measurements were taken at a calibrated camera length of 1598 mm with an X-ray energy of 11 KeV (1.12713 Å). This camera length allowed for the scattering vector  using a cone-plate geometry  with a 51 µm truncation gap. About 1 mL of Fmoc-peptide hydrogels were placed onto the lower plate to completely cover the measured area. To ensure the measurements were made in the linear viscoelastic regime, amplitude sweeps were performed at a constant frequency of 10 rad/s with shear strain 0.01–100%, where no variation in elastic modulus Primary HMFCs were obtained from ScienCell Research Laboratories. Cells were cultured as described previously . Brieflyw/w). Quantification of live cells was performed based on colorimetric method by adding MTS reagent  to the cells growing on hydrogels every 24 h. MTS (20 µL) was added to the cells and mixed gently. Further the cells were incubated for 4 h at 37 °C before reading of absorbance values at 490 nm using spectrophotometer .HMFC cells (4000 per well in 100 µL of media) were cultured on the hydrogels constitutively for 24, 48 and 72 h on the hydrogels containing peptides, Fmoc-FRGDF, Fmoc-FRGDFPHSRN, and peptide mixture of Fmoc-FRGDF/Fmoc-PHSRN  was used to stain the actin according to manufacturer’s instructions. Stained cells were observed under the fluorescent microscope . ImageJ analysis  was used to analyse the area and intensity of the staining as per the software instructions. Graph pad prism  was used to calculate the significant student t-test for the values obtained using ImageJ.w/w) was prepared. These four groups of peptides were then used to form hydrogels using a well-established pH switch method [2 backbone [Three minimalist Fmoc-SAPs, Fmoc-FRGDF, Fmoc-PHSRN and Fmoc-FRGDFPHSRN a–c were h method . Our preh method ,31. Our backbone ,33. It tHowever, when the materials were allowed to form a self-supporting hydrogel for 10 days, spectroscopic analysis of the Fmoc-SAP systems confirmed that the assembly is consistently driven by a similar mechanism, where Fmoc- interactions, stabilized by a range of hydrogen bonds in a hierarchical assembly , support−1 However, in Fmoc-PHSRN alone, there was no evidence of a β-sheet structure around 1630 cm−1. Instead, an absorption peak with a maximum at 1667 cm−1 was detected, which represents random coil structure [−1 was observed, indicating that in addition to the core of the β-sheet structure, other secondary structures predominantly composed of an element of random coil suggesting that the spatial requirements for the packing of the two peptides, particular those including proline, is not ideal. The peak at 1664 cm−1 is absent in the spectra of Fmoc-FRGDF peptide; it is suggested therefore, that conformational constraints introduced by the Fmoc-PHSRN peptide have an impact on the β-sheet based supramolecular structure formation in Fmoc-FRGDF.In each case, the FTIR and CD spectra indicate the organization of the molecules into a dominant structural ordering. Fourier transform infrared spectroscopy (FT-IR) was used to determine the dominant organization of the peptide backbone. As shown in tructure  For the Circular dichroism (CD) was used for the analysis of secondary structures of amino acids in conjunction with FT-IR. The CD spectrum b of FmocFluorescence spectroscopy has been used by many researchers to monitor the environment of the fluorenyl group ,41 Excitq−1 dependence at low q indicating elongated cylindrical structures  method to was used. As the nanostructure was determined to be fibrillar in nature from microscopy the maximum P(r) value given from the IFT calculations was attributed to the average radius of the fibrils. These calculations indicated average fibril diameters of 8.4 nm  for Fmoc-FRGDF and 6.3 nm  for Fmoc-FRGDF/Fmoc-PHSRN (r = 35.3 Å (σ = 1.2 Å) indicating a diameter of 7.1 nm, which is consistent with TEM analysis of the fibrils.Small-angle X-ray scattering (SAXS) curves supported the cylindrical nature of the nanofibrils as scattering curves for Fmoc-FRGDF, Fmoc-FRGDF/Fmoc-PHSRN, and Fmoc-FRGDFPHSRN provided a ructures a. As detoc-PHSRN b. The diG’) over corresponding viscous moduli’ (G”) of Fmoc-FRGDF, Fmoc-FRGDFPHSRN and the Fmoc-FRGDF/Fmoc-PHSRN mixture, suggesting that all three samples are viscoelastic in nature and behave as typical hydrogels. Alternatively, Fmoc-PHSRN . The structural contribution seen via spectroscopic analysis leads to a value of G’ greater than G”, indicating the structural contribution of the micelle forming properties of Fmoc-PHSRN. In order to stabilse PHSRN within the network, we utilized two strategies; combining the sequence PHSRN with Fmoc-FRGDF in a single compound to give Fmoc-FRGDFPHSRN, and the coassembly of Fmoc-FRGDF and Fmoc-PHSRN into a single assembly. Fmoc-FRGDF forms a robust, soft hydrogel with a G’ value  compared with Fmoc-FRGDFPHSRN . Surprisingly, with the addition of the nongelator peptide (Fmoc-PHSRN) to Fmoc-FRGDF in the peptide mixture, the hydrogel is quite robust with a G’ value of ~1600 Pa (50 rad/s), exceeding that of Fmoc-FRGDF alone by over two orders of magnitude, clearly demonstrating a much stronger gel than that of Fmoc-FRGDF alone.Once the broad self-assembly mechanism was understood, the mechanical properties of the samples was determined by parallel-plate rheometry, as shown in Both TEM and AFM were used to assess the nano- and microtopography of the fibers. Apart from nongelator peptide Fmoc-PHSRN, all gel-forming peptides were observed to present 5–10 nm nanofibils in diameter calculated by ImageJ, and further formed fibrillar networks a–d. In TThe AFM images revealed well-ordered networks formed in Fmoc-FRGDF e as was R), Fmoc-FRGDFPRHSRN (RP) or Fmoc-FRGDF/Fmoc-PHSRN (R + P)). HMFCs attachment and spreading on hydrogels containing R, RP and R + P were observed using actin staining at 48 h in order to allow the cells to establish and react to the various microenviroments. Cells cultured on the Fmoc-FRGDF hydrogels demonstrated viable cells, with a normal fibroblast structure. Conversely, the cells grown on Fmoc-FRGDFPHSRN were rounded, indicative of less spreading which can be attributed to lower attachment. Representative cells showed more significant spreading on the hydrogels containing a combination of Fmoc-FRGDF and Fmoc-PHSRN  were grown via seeding on the surface of the three hydrogels  can be used as an effective tool to mimic the 3D microenvironment of natural ECM, scaffolds formed via the coassembly of two different peptide signals (RGD and PHSRN) improved spreading, attachment and proliferation of cells. Overall, therefore, this work illustrates the importance of matching SAP properties to the application, and the design requirements of multicomponent self-assembled systems that can provide enhanced ECM mimicry for tissue engineering applications.


Magnaporthe oryzae were subjected to a PCR test to detect the presence/absence of avirulence (Avr) genes. Results revealed that the more frequently occurring genes were Avr-Pik (81.50%), Avr-Pita (64.16%) and Avr-Pii (47.98%), whereas the less frequently occurring genes were Avr-Pizt (19.08%) and Avr-Pia (5.20%). It was also laid out that the presence of Avr genes in M. oryzae is strongly associated with agroecosystems where the complementary resistant (R) genes exist. No significant association, however, was noted on the functional Avr genes and the major geographic locations. Furthermore, it was identified that the upland varieties locally known as “Milagrosa” and “Waray” contained all the R genes complementary to the Avr genes tested.A total of 131 contemporary and 33 reference isolates representing a number of multi-locus genotypes of  Magnaporthe oryzae, is a well-documented plant pathosystem. Valent , 10 µM each of the forward and reverse primers, 10 mM dNTP mix, 0.5 U DyNAzyme II DNA Polymerase , and sterile distilled water to a volume of 25 µL. A total of 164 isolates, from 131 newly collected and 33 old and reference isolates also collected from Philippine rice fields but maintained in Didier Tharreau’s laboratory in CIRAD, Montpellier, France was considered.Total genomic DNA was amplified using the primer sets as shown in The PCR program was set at 95 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 53–64 °C for 45 s, depending on the primer set, and 72 °C for 1 min with a final extension of 72 °C for 7 min in a PTC100  thermocycler. Polymerase chain reaction products were analyzed by 2% agarose gel electrophoresis in TAE buffer for 30 min at 100 V. Gels were stained in ethidium bromide and visualized under UV light.Multilocus genotypes (MLGs) are a unique combination of alleles across two or more loci and are useful for identifying the spread of the pathogen. Multilocus genotypes were determined through analyzing the result of the DNA sequencing from PCR products amplified utilizing the 12 microsatellite markers. Those belonging to the same MLG have the same sizes of amplified products in all of the 12 microsatellite markers using the software GeneMapper version 4.1 . The MLG:isolate ratio means the proportion of unique MLG which can be expressed as percentage from the number of isolates collected.R genes present in the rice genotype was mainly attributed to the presence of Avr genes. This is based on the gene-for-gene concept of Flor [Avr genes in the pathogen, the corresponding R genes in the host are also assumed to be present.The determination of the  of Flor  which stAVR genes in each isolate were subjected to contingency chi-square (χ2) analysis against geographic locations and types of agroecosystem utilizing the JMP-N SAS software v 10 . χ2 values computed for each cell used the formula (O − E)2 / E where O and E represent observed and expected frequencies, respectively.Data from plus/minus PCR test to assess for the presence or absence of six Avr-Pik was present in the majority of the isolates (81.50%). The relative frequencies of the other Avr genes were 64.2% (Avr-Pita), 48.0 % (Avr-Pii), 19.1% (Avr-Pizt), 8.67% (Avr-PWL3) and 5.2% (Avr-Pia). Gel electrophoresis showing the migration pattern of the different Avr genes is presented in Avr genes except for isolates PH 217 and 358, which differ in Avr-Pita. This could not be a potential error as the amplification was done three times. A comparative frequency of Avr genes found in isolates collected from each type of agroecosystem and those in the reference strains is shown in Avr-Pia and Avr-PWL3, and a very small percentage of isolates from irrigated lowland contain Avr-Pizt (Avr-Pizt .Avr genes in the isolates indicated that there was no significant association between geographic locations (islands) and the presence of Avr genes, except for Avr-PWL3, which shows to highly prevail in the Visayas , whereas the rainfed upland rice genotypes contained the highest number of R genes. Furthermore, the upland rice genotypes locally known as “Milagrosa” and “Waray” from Cavinte, Laguna possessed all of the six complementary R genes , it was similar. Avr-Pizt was exceptional because it was said to be amplified in 90% of worldwide strains and 67% of the Philippines strains (reference strains), so it seemed that our collected samples were very different. Jia et al. [Avr genes present on isolates collected in southern US from 1970 to 2009 and reported that the majority of their collections contained Avr-Pita (65.7%). The other Avr genes and their relative frequencies noted in their collections were Avr-Pizt (57.1%), Avr-Pik (11.4%), Avr-Pia (2.9%) and Avr-Pii (0.3%). In Yunnan, China, the presence of AvR-Pii was 82 out of 454 field isolates of M. oryzae [Avr-Pizt and Avr-Pik had the highest frequency (100%) and Avr1-CO39 had the lowest (2%) [PWL-2, Avr-Pii and Avr-Pizt gene-specific primer amplification had the highest frequency of 100%, 60% and 54%, respectively [Avr-Pizt. Avr-PWL3 is rarely present in M. oryzae isolates from rice [Avr-Pia in our samples.In this study, the ratio of samples amplified is similar for  samples . Avr-Piia et al.  examined. oryzae , whereasest (2%) . In Thaiectively . From thAvr-Pik and Avr-Pii occur in highest frequency in the irrigated lowland and rainfed lowland, respectively. This implies that the rice varieties planted can have significant impact on the existence of avirulence genes in the area. This result corroborates with those of Li et al. [M. oryzae. Considering the weak geographic structure of M. oryzae populations in the Philippines, where gene flow occurs among the major islands, usually through the transport of infected seeds [Avr-Pizt and AvrPii, based on our results of pathogenicity test [Significant correlations were noted on the type of agroecosystem as well as the classes of avirulence genes present. All of the cloned avirulence genes tested were present in fair proportions in the upland agroecosystem. This result reinforces the fact that rice blast is most favorable in the upland , and thai et al.  who studed seeds , the sprity test . However


To assess the effect of the implementation of a palliative care program ondo-not-resuscitate orders and intensive care unit utilization duringterminal hospitalizations.Data were retrospectively collected for all patients who died in a tertiaryhospital in Brazil from May 2014 to September 2016. We analyzed thefrequency of do-not-resuscitate orders and intensive care unit admissionsamong in-hospital deaths. Interrupted time series analyses were used toevaluate differences in trends of do-not-resuscitate orders and intensivecare unit admissions before (17 months) and after (12 months) theimplementation of a palliative care program.We analyzed 48,372 hospital admissions and 1,071 in-hospital deaths. Deathswere preceded by do-not-resuscitate orders in 276 (25.8%) cases andadmissions to the intensive care unit occurred in 814 (76%) cases.Do-not-resuscitate orders increased from 125 (20.4%) to 151 (33%) cases inthe pre-implementation and post-implementation periods, respectively (p <0.001). Intensive care unit admissions occurred in 469 (76.5%) and 345(75.3%) cases in the pre-implementation and post-implementation periods,respectively (p = 0.654). Interrupted time series analyses confirmed a trendof increased do-not-resuscitate order registrations, from an increase of0.5% per month pre-implementation to an increase of 2.9% per monthpost-implementation (p < 0.001), and demonstrated a trend of decreasedintensive care unit utilization, from an increase of 0.6% per monthpre-implementation to a decrease of -0.9% per month in thepost-implementation period (p = 0.001).The implementation of a palliative care program was associated with a trendof increased registration of do-not-resuscitate orders and a trend ofdecreased intensive care unit utilization during terminalhospitalizations. Moreover, potentially inappropriate ICUadmissions may increase the strain on the allocation of scarcecritical care resources.-11A large proportion of a population's deaths occurs inhospitals,,12 and it has been suggested that palliative care (PC)interventions can modify decisions about care goals and patientallocation.-15 For instance, advanced care planning or PC referralin the wards or during an ICU stay may reduce inappropriate ICUadmissions. It has been proposed that one of the main driversof this change is timely and sensitive communication about appropriate goals ofcare, taking into consideration the patient's condition, prognosis andvalues.There is great variability in decisions of prognostication and the limitations ofmedical treatment,-19 However, there are few studies in developingcountries describing ICU utilization during terminal hospitalizations and the impactof PC interventions in this population.-25Even though recommended by international organizations, PC availability andutilization varies widely and may be especially infrequent in developingcountries.In this study, we sought to evaluate the effect of the implementation of a PC programin trends of do-not-resuscitate (DNR) orders and ICU utilization during terminalhospitalizations. Moreover, we analyzed if this effect could be different inpatients admitted to oncological or non-oncological specialties.Hospital São Rafael (HSR).This study was approved, with a waiver for informed consent, by the Ethics Committeeof In-hospital deaths in the period from May 2014 to September 2016 (29 months) wereincluded in the study. In the case of multiple hospital admissions in the studyperiod, only the last admission was included.Hospital São Rafael is a private, not-for-profit hospitalwith 350 beds in the northeast of Brazil. Intensive care units have an openadmission policy, in which the referring physician determines ICU admission of thepatient, except in moments of scarcity of available beds, when patients may besubjected to triage.In April 2014, a flag in the electronic health record was created to identifypatients with DNR orders; however, no standard policy or PC team existed. InSeptember 2015, an institutional PC program was created and a program to increaseinstitutional awareness was initiated. The main purposes of the palliative careprogram were to promote care for all dimensions of suffering, while respecting theautonomy of patients and relatives, and to better standardize goals of care,facilitating interdisciplinary communication and identification of end-of-lifepatients. Later, in April 2016, a PC physician, together with an intensivist, anoncologist, a pediatrician, a nurse, a social worker and psychologists began roundson hospitalized patients as consultants, but not admitting patients as the primaryteam.Data on demographic and clinical variables were collected retrospectively fromelectronic health records . We alsocollected information on DNR order registrations and ICU admissions during the samehospitalization.A DNR order registration was defined as the registration of a DNR order, as per thePC program in the electronic health record. ICU admission during the samehospitalization was defined as any admission to the ICU of a patient who died in thesame hospitalization. Patients were defined as admitted to an oncological specialtyif their primary admitting team was oncology, pediatric oncology or surgicaloncology.We evaluated the effect of the implementation of a PC program on the proportion ofpatients with in-hospital deaths that had a DNR order placed and the proportion ofpatients with in-hospital deaths that had been admitted to the ICU duringhospitalization.For that purpose, we analyzed the number and proportion of DNR registrations beforeand after implementation of the PC program. As a pre-specified secondary analysis,we also analyzed the proportion of DNR registrations stratified by oncological ornon-oncological specialties. Moreover, we analyzed the number and proportion of ICUadmissions during terminal hospitalization before and after implementation of the PCprogram.Categorical variables were described as proportions. Continuous variables weredescribed as the median (interquartile range) or the mean ± standarddeviation. Proportions were evaluated with chi-square statistics. Continuousvariables were evaluated with Mann-Whitney U test or t-test. Interrupted time series analysis is aquasi-experimental design that can evaluate the effect of an intervention usinglongitudinal data series. The presence of seasonal trends wasevaluated visually and by examining the partial autocorrelation function of themodel. For all analyses, the pre-implementation phase was defined as the 17months before the implementation of the PC program (May 2014 to September 2015),and the post-implementation phase was defined as the 12 months after theimplementation of the PC program (October 2015 to September 2016).To evaluate differences in DNR orders and ICU utilization over time, we performedchi-square tests for trend. To control for secular trends, we utilizedinterrupted time series analyses using autoregressive integrated moving averagemodels, as previously described.A two-tailed p value of less than 0.05 was considered significant in all analysesperformed. Statistical analyses were performed with Statistical Package forSocial Science (SPSS), version 21.0 .From May 2014 to September 2016, there were 48,372 hospital admissions and 1071(2.2%) in-hospital deaths. Deaths were preceded by a DNR order in 276 (25.8%) cases,and an admission to the ICU occurred in 814 (76%) cases. There was a mean of 36± 7 in-hospital deaths per month in the pre-implementation phase and 38± 6 in-hospital deaths per month in the post-intervention phase (p =0.704).Description of the demographic and clinical characteristics of patients who died inthe hospital in the study period is depicted in Patients with DNR orders were younger and more frequently female, had non-elective(acute) admissions, were more often admitted to oncological specialties and wereless frequently admitted to the ICU in comparison to patients without DNR orders.Most DNR orders were placed in the wards, and patients with DNR orders were lesslikely to die in the ICU . Allpatients with DNR orders who died in the ICU  had their DNRorders placed in the ICU. However, 18 patients (28.6%) who had their DNR ordersplaced in the ICU were discharged and died in the wards.Median (IQR) time from hospital admission to DNR order registration was 5 (1 - 16)days and median (IQR) time from DNR registration to death was 4 (2 - 9) days.Additionally, there was no difference in length of hospital stay for DNR and non-DNRpatients .Characteristics of patients in the pre-implementation and post-implementation periodsare described in After the implementation of the PC program, there was an increase in the proportionof in-hospital deaths with DNR order registration from 125 (20.4%) to 151 (33%) inthe pre-implementation and post-implementation periods, respectively (p <0.001).Mean ± SD proportion of in-hospital deaths with DNR orders was 0.20 ±0.05 per month in the pre-implementation period and 0.32 ± 0.12 per month inthe post-implementation period (p = 0.037). A stepped increase in DNR orderregistrations was seen after the implementation of the PC program in September 2015, which wWe performed analyses stratified for primary admitting teams. Patients admitted tooncological specialties were younger, with a median (IQR) age of 64 (52 - 75) years,whereas patients admitted to non-oncological specialties presented with a median ageof 72 (55 - 83) years (p < 0.001). Patients admitted to oncological specialtieswere admitted to the ICU during hospitalization in 259 (55.5%) cases, and patientsadmitted to non-oncological specialties in 555 (91.9%) cases (p < 0.001).Admission to oncological specialties was associated with a lower chance of death inthe ICU when compared to patients admitted to non-oncological specialties, with 178(38.1%) and 474 (78.5%) cases, respectively.versus19 (6 - 31.5) days in patients admitted to non-oncological specialties (p <0.001).Patients admitted to oncological specialties were more likely to have DNR orders whencompared to non-oncological specialties, with 247 (52.9%) and 29 (4.8%) cases,respectively (p < 0.001). DNR orders were more likely to be placed in the wardsfor patients admitted to oncological specialties, with 191 (77.3%) cases, than inpatients admitted to non-oncological specialties, with 8 (27.6%) cases (p <0.001). DNR orders were also placed earlier for patients admitted to oncologicalspecialties, with a median of 5 (1 - 15) days from admission Of the 29 patients admitted to non-oncological specialties who had a DNR orderplaced, 19 had an acute diagnosis of sepsis, 5 were admitted for stroke, 3 forrespiratory failure and 2 for other acute diagnoses. Of those 29 patients, 5 had aprevious diagnosis of dementia, 5 were elderly frail patients, 4 presented withchronic obstructive pulmonary disease, 3 with cirrhosis, 2 with chronic kidneyfailure, 2 with heart failure, 1 did not have any other comorbidities and 7 hadother diagnoses. Those patients were admitted to internal medicine ,pneumology , neurology , general surgery ,gastroenterology , hematology , nephrology , pediatrics, orthopedics  and cardiology .The primary admitting team modified the effect of the PC program on DNR orders.Patients admitted to non-oncological specialties presented with a mean proportion ofin-hospital deaths with DNR orders of 0.01 ± 0.03 per month in thepre-implementation period and 0.10 ± 0.12 per month in thepost-implementation period (p = 0.009). On the other hand, there was no significantchange for patients admitted to oncological specialties, who presented with a meanproportion of in-hospital deaths with DNR orders of 0.48 ± 0.14 per month inthe pre-implementation period and 0.58 ± 0.14 per month in thepost-implementation period (p = 0.07).Interrupted time series analyses confirmed those results. For patients admitted tonon-oncological specialties, the pre-implementation trend was a decrease of DNRorders of -0.4% per month (95%CI = -0.6 to - 0.2), and after the implementation, thetrend was an increase of DNR orders of 2.8% per month (95%CI 2.4 to 3.2), p <0.001. For patients admitted to oncological specialties, the pre-implementationtrend was an increase of DNR orders of 1.4% per month (95%CI = 1.1 to 1.7), and thepost-implementation trend was not significantly different, with an increase of 2.6%per month (95%CI = 2.1 to 3.1), p = 0.149.Overall, ICU admission during hospitalization occurred in 469 (76.5%) patients in thepre-implementation period and in 345 (75.3%) patients in the post-implementationperiod (p = 0.654) . There wThe mean ± SD of in-hospital deaths with ICU admission during thehospitalization was 0.76 ± 0.04 per month in the pre-implementation periodand did not change in the post-implementation period, in which the proportion was0.76 ± 0.07 per month (p = 0.778).Interrupted time series analyses, however, demonstrated a change in the slope of thetrend of ICU admission. In the pre-implementation phase, there was an increase of0.6% per month (95%CI = 0.5 to 0.7) of ICU admissions among in-hospital deaths. After tIn the present study, the implementation of a PC program was associated with anincreased trend in prescription of DNR orders, especially in patients admitted tonon-oncological specialties, and a decreased trend in ICU utilization duringterminal hospitalizations. However, there was a high rate of intensive care unitutilization in patients dying in the hospital, even though patients with DNR orderswere less likely to die in the ICU. Because hospitals are a major site for end-of-lifecare,-4 interventions to improve PC during terminalhospitalizations have been implemented and analyzed in theliterature.-15Palliative care has been suggested to improve communication, definitions ofgoals-of-care and to alleviate distressing symptoms in terminal phases ofdiseases.,29 For example, in a systematic review, it wasdemonstrated that PC interventions are associated with a reduction of 37% in thechance of ICU admission. Nevertheless, in the present study, the rate of ICUadmission during terminal hospitalization and death in the ICU were high, at 76% and61%, respectively. Even among DNR patients, ICU admission and death in the ICUapproached 42% and 16%, respectively. Although high, our results are not verydifferent from previous studies, which demonstrate that almost one third of patientswith serious illnesses die in the ICU or with ventilatorsupport.Our results are aligned with the literature on the impact of PC interventions, inwhich PC has been associated with modifications in goals-of-care definitions andresource utilization.,30,31 It has beenhypothesized that some of this difference may be due to misunderstandings about thetrajectory of the disease or lack of familiarity with PC by theproviders. It is possible that the implementation of theprogram had a greater effect in this more vulnerable population of non-cancerpatients, as cancer patients may have already been receiving more appropriateend-of-life care, even though this correlation should be carefully examined.Our data demonstrated that the implementation of a PC program increased DNR orders inpatients admitted to non-oncological specialties but had no effect on patientsadmitted to oncological specialties. It has been shown before that the diagnosis ofcancer influences end-of-life care, with cancer patients receiving, generally, lessaggressive care and better quality of end-of-life care.,33 Moreover, to our knowledge, this study is the firstto demonstrate that a hospital PC program had an impact on outcomes in Brazil, eventhough it is a country in which it has been shown that quality of end-of-life caremay be poor and PC of limited access. Additionally, it has been suggestedthat there are differences between Brazil and other countries in things people seemto give importance to at the end of life, such as the setting ofdeath, with Brazilians valuing more "living as long aspossible" and being more likely to die in the hospital.Our study was one of the few to analyze ICU utilization during terminalhospitalizations and to analyze a PC intervention in a developingcountry. This method of analysis allows for theinvestigation of potential biases that are common in implementation studies, such asthe secular trend bias, in which the outcome may be increasing or decreasing withtime, irrespective of the intervention implemented.Another strength of this study is that we utilized interrupted time series analysesto evaluate the trends of DNR orders and ICU admissions. Interrupted time seriesanalysis is a robust method, which has been considered the "next best approach fordealing with interventions when randomization is not possible or clinical data isnot available".However, our study has some limitations. This study was performed in only one center,so its generalizability may be reduced. Nevertheless, we have analyzed a largenumber of in-hospital deaths for 29 months, and our results may be generalized toother similar settings. This study was also retrospective and based on electronichealth record analyses, which may be subject to biases. Another limitation is thatwe were not able to retrieve more specific information on the quality of end-of-lifecare of the patients enrolled.The implementation of a palliative care program was associated with a trend ofincreased registration of do-not-resuscitate orders and a decrease of intensive careunit utilization during terminal hospitalizations. However, the increasedregistration of do-not-resuscitate orders after the implementation was seen inpatients admitted to non-oncological specialties but not in patients admitted tooncological specialties.JGR Ramos and FC Tourinho contributed to the design, acquisition, analysis andinterpretation of data and the drafting and revising of the manuscript. PBorrione, PBP Batista and AV Mendes contributed to the analysis andinterpretation of data and the drafting and revising of the manuscript. P Azi, VCosta, T Andrade and Z Reis contributed to the conception of the study andcritical revision of the manuscript. All authors have approved the final versionof the manuscript.


Dinophysis acuminata occur every year in an important mussel cultivation area in Port Underwood, Marlborough Sounds, New Zealand. Annual maximum cell numbers range from 1500–75,000 cells L−1 and over 25 years of weekly monitoring the D. acuminata bloom has never failed to exhibit peaks in abundance at some time between spring and autumn. During winter (June–August) the dinoflagellate is often undetectable, or at low levels (≤100 cells L−1), and the risk of diarrhetic shellfish poisoning (DSP)-toxin contamination over this period is negligible. Bloom occurrence may be coupled to the abundance of D. acuminata prey (Mesodinium sp.) but the mechanism by which it maintains its long-term residence in this hydrologically dynamic environment is unknown. The toxin profile of D. acuminata is dominated by pectenotoxin-2 (PTX-2) and dinophysistoxin-1 (DTX-1), but the cellular toxin content is low. It is rare that free DTX-1 is detected in mussels as this is invariably exclusively present as fatty acid-esters. In only five out of >2500 mussel samples over 16 years have the levels of total DTX-1 marginally exceeded the regulated level of 0.16 mg kg−1. It is also rare that free PTX-2 is detected in mussels, as it is generally only present in its hydrolysed non-toxic PTX-2 seco acid form. The D. acuminata alert level of 1000 cells L−1 is often exceeded without DTX-1 residues increasing appreciably, and this level is considered too conservative.Blooms of the dinoflagellate  Dinophysis (and some benthic Prorocentrum spp.) has become a significant quality assurance issue for shellfish aquaculture worldwide. Dinophysis spp. produce a suite of lipophilic polyether secondary metabolites within the okadaic acid  and pectenotoxin (PTX-1–PTX-11) families. These toxins are internationally regulated at a maximum permissible level of <0.16 mg kg−1 [Since the phenomenon was first identified ,2, diarr mg kg−1  and ther2 inlet on the north-east coast of the South Island, New Zealand  growing regions of the Marlborough Sounds. The high productivity of the inlet (approximately 8000 tonnes of mussels per annum) is attributed to the fertilising effects of local wind-induced upwelling and its proximity to the Wairau River out-welling plume. Since weekly toxic phytoplankton and marine biotoxin monitoring began in Port Underwood in the early 1990s, it has become apparent that the inlet has a resident population of Dinophysis acuminata that blooms for short periods every year between spring (September–October) and autumn (March–April).Port Underwood is a 24 km Zealand , and is Dinophysis acuta and Dinophysis acuminata from various locations around the South Island coast of New Zealand has been described [D. acuminata cells sampled from Port Underwood at various times showed low levels of okadaic acid (trace–0.4 pg/cell) and dinophysistoxin-1 (DTX-1) (0.1–0.5 pg cell−1) but much higher levels of pectenotoxin-2 (PTX-2) (2.4–16.9 pg cell−1). The cellular content of OA and its esters in D. acuta was 33 times higher and for PTX-2 and PTX-11, 8–42 times higher, respectively, than in D. acuminata. Only about 6% of total PTX-2 was present as the PTX-2 seco acid in D. acuminata cells. The toxin content of D. acuminata cells from Port Underwood was significantly lower than that of the same species from elsewhere in the South Island (Akaroa Harbour). DTX-1 was not detected in any D. acuta cells and DTX-2 was not detected in either species. A similarly low toxin content (0.01–1.8 pg OA + DTX-1 cell−1) has been observed in D. acuminata cells from the north-western Atlantic [The cellular toxin content of the major pectenotoxin and okadaic acid group toxins in escribed . D. acumDinophysis species throughout the world [−1 okadaic acid equivalents that must not be exceeded in the edible portion of the shellfish. Okadaic acid group toxins  and pectenotoxins  are considered additive and, above this level, shellfish harvesting is prohibited. Pectenotoxin-seco acid analogues  are not considered a hazard and there is no non-permissible level.Pectenotoxins are the predominant polyether macrolides found in the he world . At leashe world . Pectenohe world . Subsequhe world  that PTXhe world . This enhe world . In the he world , an evalhe world . This stD. acuminata ecology and the environmental circumstances accompanying dinophysis-toxin contamination events.The data presented here, accumulated over 25 years of weekly monitoring of phytoplankton and shellfish, provide a unique perspective on the magnitude of the DSP-toxin contamination problem in Port Underwood. Additional data from the occasional opportunistic sampling of the water column within the inlet illuminate some aspects of D. acuminata were on average (n = 10) 41.3 ± 2.7 µm (cell length) by 27.6 ± 1.6 µm (cell width) and, in terms of cell size and general morphology , deeply red-pigmented cells that were most commonly observed during the early phases of bloom development D It was sp. prey  and thatD. acuminata abundance occurred at some time between spring and autumn every year from 1994 to 2018 (−1. Mid-winter (June–August) was the period when cells were most likely to be absent from the plankton  and the phototrophic ciliate Mesodinium sp. A significant proportion of the D. acuminata populations at these depths (14% at 12 m and 23% at 15 m) were comprised of the large, swollen, red-pigmented cells described previously  but outside of the main phytoplankton bloom periods represented by high chlorophyll a concentrations  was carried out (D. acuminata cells (On 14 occasions during ried out . The dista cells .−1) in the majority  of samples analysed at both sites. At the Horahora site, 22.6% of samples had PTX-2sa above the reporting level. The maximum concentrations of PTX-2sa observed at Whangakoko, Opihi, and Horahora were 1.7 mg kg−1 (March 2013), 1.4 mg kg−1 (April 2005), and 0.24 mg kg−1 (October 2009), respectively. At Whangakoko, parent PTX-2 was only above the reporting level in 27 out of 860 samples analysed (3.1%), with a maximum concentration of 0.039 mg kg−1 in April 2016. At Opihi, only 1.1% of samples contained reportable levels of PTX-2, with a maximum concentration of 0.025 mg kg−1 (January 2012). At Horahora, free PTX-2 was not detected. It is likely that the concentrations of PTX-2sa were, in fact, significantly higher than those reported here, since PTX-2sa-esters were not included in these analyses. According to Torgeston et al. [Mytilus edulis) may be in the form of fatty acid esters, with 16:0 and 14:0 predominant. Likewise, Blanco et al. [Mesodesma donacium. They also found that PTX-2 and PTX-2sa were more rapidly eliminated from the digestive gland than PTX-2sa esters.A summary of the results of the weekly analysis of mussel tissues from the Whangakoko and Opihi monitoring sites  shows thn et al. , >80% of−1 at Whangakoko on 3 March 2010. Between October 2003  and July 2018, 2307 samples were screened for the total DTX-1. Of these, only 11 samples (0.4%) had a total DTX-1 above 0.1 mg kg−1 and only 5 samples (0.2%) had concentrations at or above the regulatory level of 0.16 mg kg−1 . Abal et al. [−1 from five to nine per 2307 samples analysed (0.2–0.3%).Only a small proportion of samples between 2002 and 2018 had concentrations of total DTX-1 above the reporting level . DTX-1 w mg kg−1 . The cell et al.  have recD. acuminata that, over 24 years of continuous weekly monitoring, has never failed to bloom to some degree, at some time, from early spring to late autumn. Blooms developed in near-bottom waters in the most inland reaches of the inlet. It is believed that the abundance of Mesodinium sp. prey at depth may be an important precursor of the blooms. Mesodinium sp. is common in the inlet but cells are poorly preserved in Lugol’s iodine and numbers were not recorded during routine monitoring, so no definitive data exists to demonstrate the relationship between populations of prey and predators. D. acuminata numbers rarely exceed 10,000 cells L−1 but low numbers of cells are present in the water column throughout much of the year. Cultivated mussels show evidence of this in the high proportion of samples that show low concentrations of (non-regulated) PTX-2sa in their flesh. The DTX-1 content of D. acuminata cells is low and only a small proportion of mussel samples show evidence of DTX-1 accumulation (primarily as DTX-1 fatty acid-esters) above the limit of reporting. Over 16 years of weekly LC-MS analysis, only a small proportion of mussel samples (0.2%) achieved or marginally exceeded the regulated level of 0.16 mg kg−1. In every case when this level was reached, D. acuminata cell numbers exceeded 10,000 cells L−1. Cell numbers of up to 5000 cells L−1 did not result in toxicity exceeding the regulatory limit, and the current action level of 1000 cells L−1 could be reviewed. It has been shown in cultured and natural populations of Dinophysis spp. [D. acuminata but the long-term data set presented here clearly shows that, given the inherent low toxicity of this population, any natural fluctuations in toxin quota are unlikely to be of any practical significance with respect to the toxin content of cultivated mussels. Increasing concentrations of PTX-2sa are a good indicator of the imminence of DTX-1 contamination and the addition of PTX-2sa ester quantification to the routine monitoring protocol would likely increase the sensitivity of this indicator.Port Underwood has a resident population of sis spp. ,27 that Sea-water samples were collected weekly at three sites  in Port Underwood  with a tTM mussels  were collected weekly from mussel culture long-lines at the same sites as the phytoplankton samples. Routinely, samples were collected from a depth of 6 m, although on some occasions during significant bloom events, additional samples were also collected from near the surface (top) and from a depth of 12 m (bottom). Phytoplankton and shellfish samples were couriered to the Cawthron phytoplankton and biotoxin laboratories and the results of the analyses were available within 24 hours of receipt of the samples.Samples of GreenshellPrior to 2002, diarrhetic shellfish poisoning (DSP) toxicity of shellfish samples was analysed using the standard mouse bioassay . HoweverFrom 2002, all mussel samples were analysed using the multi-residue LC-MS/MS method for lipophilic algal toxins developed and validated by McNabb et al. ,22. Shel−1, respectively. From 2016 onwards, the limit of reporting of the total DTX-1 was also reduced to <0.01 mg kg−1.Until 2016, the lower limits of reporting of PTX-2, PTX-2 seco acid, and total DTX-1, the only lipophilic toxin residues of any significance in these shellfish, were <0.01, <0.01, and <0.05 mg kg


Several studies have already identified the prognostic markers in colorectal cancer (CRC) based on somatic copy number alteration (SCNA). However, very little information is available regarding their value as a prognostic marker. Gene dosage effect is one important mechanism of copy number and dosage-sensitive genes are more likely to behave like driver genes. In this work, we propose a new pipeline to identify the dosage-sensitive prognostic genes in CRC. The RNAseq data, the somatic copy number of CRC from TCGA were assayed to screen out the SCNAs. Wilcoxon rank-sum test was used to identify the differentially expressed genes in alteration samples with |SCNA| > 0.3. Cox-regression was used to find the candidate prognostic genes. An iterative algorithm was built to identify the stable prognostic genes. Finally, the Pearson correlation coefficient was calculated between gene expression and SCNA as the dosage effect score. The cell line data from CCLE was used to test the consistency of the dosage effect. The differential co-expression network was built to discover their function in CRC. A total of six amplified genes  were found to be associated with poor prognosis. They demonstrate a stable prognostic classification in more than 50% threshold of SCNA. The average dosage effect score was 0.5918 ± 0.066, 0.5978 ± 0.082 in TCGA and CCLE, respectively. They also show great stability in different data sets. In the differential co-expression network, these six genes have the top degree and are connected to the driver and tumor suppressor genes. Function enrichment analysis revealed that gene NDUFB4 and GTF2E1 affect cancer-related functions such as transmembrane transport and transformation factors. In conclusion, the pipeline for identifying the prognostic dosage-sensitive genes in CRC was proved to be stable and reliable. Studieshe world . SCNA gehe world .vice versa, this gene would be defined as dosage-sensitive gene. With respect to the unstable and complex nature of expression regulation, the DNA copy number is relatively more stable. Therefore, the copy number of dose-sensitive genes is more likely to be used as a driver gene in cancer. Some of the dosage-sensitive genes (DSGs) such as CD274/PD-L1 gene amplification  is pathogenic  were identified as copy number amplification samples (CNAS), the samples with SCNA < = −x were identified as copy number deleted samples (CNDS), and the samples with | SCNA | < x were identified as copy number non-altered samples (CNNS). The location information of chromosomes was obtained from the HGNC database (http://genome.ucsc.edu/), and more than 80% of genes with 0 value were filtered out. The test data-set was collected from the Cancer Cell Line Encyclopedia .The data of CNA, RNA-seq data, and clinical data of CRC were downloaded from the TCGA database. By mapping the copy number probe across the reference genome of hg38, the SCNA at gene level was calculated using Gistic2 software . The valdatabase . RNAseq PSGs of SCNA were screened in five steps as described below:p-value was corrected by the Benjamini-Hochberg method. As there were very small differences in gene expression between SCNA and CNNS samples their false discovery rate (FDR) < 0.1 and p < 0.01, fold change >1.2 were considered as differential expression.Step 1: Set x (x > 0) as the threshold for SCNA, then the samples of CRC were classified into three groups, somatic copy number amplification samples (CNAS), somatic copy number deletions samples (CNDS), and somatic copy number non-alteration samples (CNNS). The number of CNAS or CNDS was more than or equal to 10. Wilcoxon rank-sum test was performed to identify differentially expressed genes between CNAS and CNNS and between CNDS and CNNS. The p-value < 0.01 and FC > 1.2) and copy number amplification (SCNA > x) in CNAS, and the genes with expression down-regulation  and copy number deletion (SCNA < −x) in CNDS as candidates for the dosage-sensitive gene.Step 2: In order to further screen the candidate genes on the basis of Step 1. We identified genes with expression up-regulation .Step 5: In order to further screen dosage-sensitive genes from stable PSGs in different SCNA threshold, the prognostic sensitive abnormal genes of DSGs were selected. Linear regression was applied to assess the dosage-sensitivity. The R-value represents the dosage-effect score. The genes with the In order to verify the stability of the dosage-sensitivity of PDSGs, the correlation coefficients between gene expression and copy number alteration were calculated with the RNA-seq of CRC and CNA at gene level downloaded from the CCLE database. These values were compared with the findings obtained from TCGA.In order to further identify the genes affected by PDSGs, Pearson correlation coefficients of these six PDSGs and other genes was calculated as co-expression values in CNAS or CNDS, CNNS. Gene pairs with correlation coefficients higher than 0.5 in one group and less than 0.1 in another group were screened as differentially co-expressing gene pairs. Network visualization tools were executed using Cytoscape .All the analysis was performed in the R computing environment. Survival curves were estimated using the Kaplan-Meier method. Gene function enrichment was performed using the Cluster Profiler package .A total of 448 CRC samples with SCNA and RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA). The samples were screened for survival information. There were 22,752 genes, of these 17,442 were protein-coding and 14,688 were differentially expressed.After applying FDR < 0.1 and FC > 1.2, 6,814 genes had up-regulated expression in CNAS. Twenty-five genes had a down-regulated expression in CNDS. Cox regression analysis was applied to calculate the correlation between SCNA and survival time. A total of 215 prognosis-sensitive genes (PSGs) significantly related to SCNA were obtained, of these 214 were amplified and one was deleted. Next, the 21 SCNA threshold value was raised from 0.1 to 0.5 at a step of 0.02. For each threshold, the samples were classified into CNNS, CNAS, CNDS group and logRank test between CNNS and CNAS, CNDS and CNNS was performed. As shown in After further screening stable PSGs which are highly affected by copy number dosage effect, the Pearson correlation coefficient between copy number and corresponding expression value (FPKM) of these 15 genes was calculated. Finally, six genes  which are stable PSGs Figure 2p-value = 0.087199 and p-value = 0.12643) in 0.5 SCNA threshold was not significant, their classification curves were distinctly separated. The non-significance can be primarily attributed to the very small number of samples with SCNA threshold >0.5.Kaplan-Meier survival curve analysis revealed six (6) PDSGs with similar results in a different threshold of SCNA. In the 0.1 SCNA threshold value, genes GTF2E1, NDUFB4, IQCB1, KPNA 1 and WDR5B had a significant classification effect . As a result, FRA3D (3q25.32) and FRA3C were found to be near to six PDSGs. Correlation analysis of SCNA in six PDSGs and the genes in FRA3D and FRA3C was performed. Gene RSRC1 (R = 0.82), MLF1(R = 0.82) in FRA4D, and LPP (R = 0.80) in FRA3C had lowest relationship with PDSGs. Thus we infer that the breakpoints in fragile site may explain the reason for the nearby region and a similar SCNA value.Research have shown that heterogeneity of copy number alterations exists in ongoing unstable chromosome in COAD . There aR) > 0.5 and (R) < 0.1 in a different class of samples by calculating the differences of gene co-expression between CNAS and CNNS. A total of 234 co-expressed gene pairs were observed and 215 genes (R > 0.5), but not co-expressed in non-alteration samples (R < 0.1), while the other 40 pairs behaved in a reverse manner. In the network, gene NDUFB4, SEC22A had the highest degree (109 and 45 respectively) consisting of 15 co-linked genes. The genes CAPN14 and CMPK2 were affected by three PDSGs . This suggests that PDSGs are closely linked and interact with each other.In order to further explore if these six PDSGs can also affect the expression of other genes in CRC, we screened genes with (R = 0.59). WNT8B one member of the WNT signal was differentially expressed in COAD (http://bioinfo.mc.vanderbilt.edu/TSGene/) revealed 16 TSGs .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.